Defective regulation of epithelial secretion underlies diseases ranging from cystic fibrosis to secretory diarrhea. This project has established the importance of a basolateral transporter (the Na-K-2CI cotransporter NKCC1 ) in the control of epithelial CI secretion and identified a novel regulatory mechanism involving alterations in NKCC1 surface expression mediated by protein kinase C (PKC). Experiments are now proposed to map endocytic pathways of NKCC1 and to delineate the role of specific PKC isoforms in NKCC1 internalization. New data suggest that PKC-mediated endocytic internalization of NKCC1 (and, possibly, exocytic insertion of the Na-HCO3 cotransporter NBC1) may account in part for the reciprocal relationship between CI and HCO3 transport in various secretory epithelia. The potential role of Rho-family GTPase members in PKC-directed endocytosis, and in membrane/cytoskeletal remodeling will be explored. The novel PKC isoforms that mediate NKCC1 internalization also appear to play a role in lamellipodia formation and cell extension during spreading and migration. Speculatively, this general action of novel PKCs may represent a means to rapidly redeploy basolateral ion transporters to, or away from, the leading edge of epithelial cells, a recently-recognized characteristic important in epithelial cell migration and wound closure. These studies utilize epithelial cell lines, biochemical and genetic techniques, and confocal microscopy to address these several issues. In Aim 1, NKCC1 surface dynamics will be examined in response to selected PKC activators and inhibitors, and NBC1 surface dynamics will be examined during Ca v2- and CO2-stimulated HCO3 secretion. In Aim 2, the endocytic pathway of NKCC1 will be defined using functional and morphologic criteria, aided by the use of an EGFP-NKCC1 construct. Endocytosis signals in NKCC1 and the factors that determine the fate of endocytosed NKCC1 will be examined. Finally, the endocytic redistribution of NKCC1 and NBC1 in response to epithelial spreading, and the possible role of PKCs, PKCS, and Rho-family GTPase targets in the associated membrane-cytoskeletal remodeling, will be examined. The regulation of plasma membrane composition and plasticity by novel PKC isoenzymes forms a common thread linking these studies of the endocytic regulation of basolateral salt transporters to an emerging line of investigation involving membrane dynamics during epithelial repair.